Characterize the fetal kidney reference from the kidney cell atlas
Maud PLASCHKA
2024-08-07
Introduction
The aim is to characterize the human fetal kidney from the kidney
cell atlas. You can find more about the human kidney atlas here: https://www.kidneycellatlas.org/ [1] The rds data can be
download using the download link: https://datasets.cellxgene.cziscience.com/40ebb8e4-1a25-4a33-b8ff-02d1156e4e9b.rds.
The reference was downloaded and created in the script
scripts/prepare-fetal-references.R.
Packages
library(Seurat)
library(SCpubr)
library(tidyverse)
library(patchwork)
set.seed(params$seed)
options(future.globals.maxSize = 891289600000)Base directories
# The base path for the OpenScPCA repository, found by its (hidden) .git directory
repository_base <- rprojroot::find_root(rprojroot::is_git_root)
# The path to this module
module_base <- file.path(repository_base, "analyses", "cell-type-wilms-tumor-06")Input files
The input file ../scratch/fetal_kidney.rds is the output of the
script prepare-fetal-references.R.
Output file
We will save the result of the differential expression analysis in
results/references/00b_marker_genes_fetal_kidney_Stewart.csv.
Notebook is saved in the notebook/00-reference
directory.
path_to_output <- file.path(module_base, "results", "references")Analysis
Load the reference
fetal_kidney <- readRDS(params$fetal_kidney_path)
d1 <- do_DimPlot(fetal_kidney, reduction = "umap", dims = c(1, 2), group.by = "compartment", label = TRUE, repel = TRUE) + NoLegend()
d2 <- do_DimPlot(fetal_kidney, reduction = "umap", dims = c(1, 2), group.by = "cell_type", label = TRUE, repel = TRUE) + NoLegend()
d1 | d2
Characterization of compartment and cell types in the reference
Here, we use an unbiased approach to find transcripts that characterized the different compartments and cell types.
This is just to get markers genes of the different population, in case some could be of interest for the Wilms tumor annotations.
We run DElegate::FindAllMarkers2() to find markers of
the different clusters and manually check if they do make sense.
DElegate::FindAllMarkers2() is an improved version of
Seurat::FindAllMarkers() based on pseudobulk differential
expression method. Please check the preprint from Hafemeister and
Halbritter: https://www.biorxiv.org/content/10.1101/2023.03.28.534443v2
and tool described here: https://github.com/cancerbits/DElegate
Find marker genes for each of the compartment
de_results <- DElegate::FindAllMarkers2(fetal_kidney, group_column = "compartment", )## Warning in size + sum(size_args, na.rm = FALSE): NAs produced by integer
## overflow# filter the most relevant markers
s.markers <- de_results[de_results$padj < params$padj_threshold & de_results$log_fc > params$lfc_threshold & de_results$rate1 > params$rate1_threshold, ]
DT::datatable(s.markers,
caption = ("marker genes"),
extensions = "Buttons",
options = list(
dom = "Bfrtip",
buttons = c("csv", "excel")
)
)# Select top 5 genes for heatmap plotting
s.markers <- na.omit(s.markers)
s.markers %>%
group_by(group1) %>%
top_n(n = 5, wt = log_fc) -> top5
# subset for plotting
Idents(fetal_kidney) <- fetal_kidney$compartment
cells <- WhichCells(fetal_kidney, downsample = 100)
ss <- subset(fetal_kidney, cells = cells)
ss <- ScaleData(ss, features = top5$feature)
p1 <- SCpubr::do_DimPlot(fetal_kidney, reduction = "umap", group.by = "compartment", label = TRUE, repel = TRUE) + ggtitle("compartment")
p2 <- DoHeatmap(ss, features = top5$feature, cells = cells, group.by = "compartment") + NoLegend() +
scale_fill_gradientn(colors = c("#01665e", "#35978f", "darkslategray3", "#f7f7f7", "#fee391", "#fec44f", "#F9AD03"))
p3 <- ggplot(fetal_kidney@meta.data, aes(compartment, fill = compartment)) +
geom_bar() +
NoLegend()
common_title <- sprintf("Unsupervised clustering %s, %d cells", fetal_kidney@meta.data$orig.ident[1], ncol(fetal_kidney))
show((((p1 / p3) + plot_layout(heights = c(3, 2)) | p2)) + plot_layout(widths = c(1, 2)) + plot_layout(heights = c(3, 1)) + plot_annotation(title = common_title))
write_csv(de_results, file = file.path(path_to_output, "00a_marker_compartment_fetal_kidney_Stewart.csv"))Find marker genes for each of the cell types
de_results <- DElegate::FindAllMarkers2(fetal_kidney, group_column = "cell_type")## Warning in size + sum(size_args, na.rm = FALSE): NAs produced by integer
## overflow# filter the most relevant markers
s.markers <- de_results[de_results$padj < params$padj_threshold & de_results$log_fc > params$lfc_threshold & de_results$rate1 > params$rate1_threshold, ]
DT::datatable(s.markers,
caption = ("marker genes"),
extensions = "Buttons",
options = list(
dom = "Bfrtip",
buttons = c("csv", "excel")
)
)# Select top 5 genes for heatmap plotting
s.markers <- na.omit(s.markers)
s.markers %>%
group_by(group1) %>%
top_n(n = 5, wt = log_fc) -> top5
# subset for plotting
Idents(fetal_kidney) <- fetal_kidney$cell_type
cells <- WhichCells(fetal_kidney, downsample = 100)
ss <- subset(fetal_kidney, cells = cells)
ss <- ScaleData(ss, features = top5$feature)
p1 <- SCpubr::do_DimPlot(fetal_kidney, reduction = "umap", group.by = "cell_type", label = TRUE, repel = TRUE) + ggtitle("cell_type") + NoLegend()
p2 <- DoHeatmap(ss, features = top5$feature, cells = cells, group.by = "cell_type") + NoLegend() +
scale_fill_gradientn(colors = c("#01665e", "#35978f", "darkslategray3", "#f7f7f7", "#fee391", "#fec44f", "#F9AD03"))
p3 <- ggplot(fetal_kidney@meta.data, aes(cell_type, fill = cell_type)) +
geom_bar() +
NoLegend() +
scale_x_discrete(guide = guide_axis(angle = 90))
common_title <- sprintf("Unsupervised clustering %s, %d cells", fetal_kidney@meta.data$orig.ident[1], ncol(fetal_kidney))
show((((p1 / p3) + plot_layout(heights = c(3, 2)) | p2)) + plot_layout(widths = c(1, 1)) + plot_layout(heights = c(3, 1)) + plot_annotation(title = common_title))
write_csv(de_results, file = file.path(path_to_output, "00a_marker_cell-type_fetal_kidney_Stewart.csv"))References
Session info
sessionInfo()## R version 4.4.0 (2024-04-24)
## Platform: aarch64-apple-darwin20
## Running under: macOS 15.1
##
## Matrix products: default
## BLAS: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRblas.0.dylib
## LAPACK: /Library/Frameworks/R.framework/Versions/4.4-arm64/Resources/lib/libRlapack.dylib; LAPACK version 3.12.0
##
## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
##
## time zone: America/New_York
## tzcode source: internal
##
## attached base packages:
## [1] stats graphics grDevices utils datasets methods base
##
## other attached packages:
## [1] patchwork_1.2.0 lubridate_1.9.3 forcats_1.0.0 stringr_1.5.1
## [5] dplyr_1.1.4 purrr_1.0.2 readr_2.1.5 tidyr_1.3.1
## [9] tibble_3.2.1 ggplot2_3.5.1 tidyverse_2.0.0 SCpubr_2.0.2
## [13] Seurat_5.1.0 SeuratObject_5.0.2 sp_2.1-4
##
## loaded via a namespace (and not attached):
## [1] RColorBrewer_1.1-3 rstudioapi_0.16.0 jsonlite_1.8.8
## [4] magrittr_2.0.3 spatstat.utils_3.1-0 farver_2.1.2
## [7] rmarkdown_2.28 DElegate_1.2.1 fs_1.6.4
## [10] vctrs_0.6.5 ROCR_1.0-11 spatstat.explore_3.3-2
## [13] htmltools_0.5.8.1 gridGraphics_0.5-1 sass_0.4.9
## [16] sctransform_0.4.1 parallelly_1.38.0 KernSmooth_2.23-24
## [19] bslib_0.8.0 htmlwidgets_1.6.4 ica_1.0-3
## [22] plyr_1.8.9 plotly_4.10.4 zoo_1.8-12
## [25] cachem_1.1.0 igraph_2.0.3 mime_0.12
## [28] lifecycle_1.0.4 pkgconfig_2.0.3 Matrix_1.7-0
## [31] R6_2.5.1 fastmap_1.2.0 MatrixGenerics_1.16.0
## [34] fitdistrplus_1.2-1 future_1.34.0 shiny_1.9.1
## [37] digest_0.6.37 colorspace_2.1-1 rprojroot_2.0.4
## [40] tensor_1.5 RSpectra_0.16-2 irlba_2.3.5.1
## [43] crosstalk_1.2.1 labeling_0.4.3 progressr_0.14.0
## [46] fansi_1.0.6 spatstat.sparse_3.1-0 timechange_0.3.0
## [49] httr_1.4.7 polyclip_1.10-7 abind_1.4-5
## [52] compiler_4.4.0 bit64_4.0.5 withr_3.0.1
## [55] viridis_0.6.5 fastDummies_1.7.4 highr_0.11
## [58] MASS_7.3-61 tools_4.4.0 lmtest_0.9-40
## [61] httpuv_1.6.15 future.apply_1.11.2 goftest_1.2-3
## [64] glue_1.7.0 nlme_3.1-166 promises_1.3.0
## [67] grid_4.4.0 Rtsne_0.17 cluster_2.1.6
## [70] reshape2_1.4.4 generics_0.1.3 gtable_0.3.5
## [73] spatstat.data_3.1-2 tzdb_0.4.0 data.table_1.16.0
## [76] hms_1.1.3 utf8_1.2.4 spatstat.geom_3.3-2
## [79] RcppAnnoy_0.0.22 ggrepel_0.9.5 RANN_2.6.2
## [82] pillar_1.9.0 vroom_1.6.5 limma_3.60.4
## [85] yulab.utils_0.1.7 spam_2.10-0 RcppHNSW_0.6.0
## [88] later_1.3.2 splines_4.4.0 lattice_0.22-6
## [91] bit_4.0.5 survival_3.7-0 deldir_2.0-4
## [94] tidyselect_1.2.1 locfit_1.5-9.10 miniUI_0.1.1.1
## [97] pbapply_1.7-2 knitr_1.48 gridExtra_2.3
## [100] edgeR_4.2.1 scattermore_1.2 xfun_0.47
## [103] statmod_1.5.0 matrixStats_1.3.0 DT_0.33
## [106] stringi_1.8.4 lazyeval_0.2.2 yaml_2.3.10
## [109] evaluate_0.24.0 codetools_0.2-20 ggplotify_0.1.2
## [112] cli_3.6.3 uwot_0.2.2 xtable_1.8-4
## [115] reticulate_1.38.0 munsell_0.5.1 jquerylib_0.1.4
## [118] Rcpp_1.0.13 globals_0.16.3 spatstat.random_3.3-1
## [121] png_0.1-8 spatstat.univar_3.0-0 parallel_4.4.0
## [124] assertthat_0.2.1 dotCall64_1.1-1 sparseMatrixStats_1.16.0
## [127] listenv_0.9.1 viridisLite_0.4.2 scales_1.3.0
## [130] ggridges_0.5.6 crayon_1.5.3 leiden_0.4.3.1
## [133] rlang_1.1.4 cowplot_1.1.3